Gentjan Hykaj, Anila Hoda, Ylli Bišoku and Lumturi Papa

Abstract

Goats play an
important role for the Albanian farmer community of marginal area. Muzhake is
one of local goat breeds distributed in Southeastern part of Albania. Genetic
characterization of native breeds is very important in conservation strategy
designing. The aim of the present study was to estimate the genetic variability
of Muzhake goat breed using microsatellite markers. The genomic DNA from 30
unrelated individuals, was analyzed by typing 30 microsatellite markers. Allele
diversity, observed and expected heterozygosities, inbreeding coefficient were
calculated. A total of 240 alleles were distinguished. All the microsatellites
were highly polymorphic, with mean allelic number of 8, ranging 4-18 per locus.

The observed
heterozygosity ranged between 0.33 to 0.93, with mean value of 0.69. PIC values
ranged from 0.39 to 0.88, with mean of 0.71. It was noticed a low rate of
inbreeding within breed (FIS = 0.07). The effective number of alleles
varied from 1.47 to 11.5 with a mean 4.69. The value of Shannon information
index (I) ranges from 0.77 to 2.49. A bottleneck analysis indicated no
bottleneck in Muzhake breed. The population was not in Hardy-Weinberg
equilibrium (HWE) for 9 out 30 loci. The results of the study indicate very high
level of gene diversity. Most of the loci showed significant deviation from HWE,
probably due to Wahlund effect. The set of used markers was highly informative.
The results provided here may be useful in developing a national plan and
strategy for the conservation of this breed.

Genetic diversity of Albanian
local goat breeds was estimated previously using microsatellite markers (Hoda et
al 2011). Muzhake is an important local goat breed, located at south and
south-east of Albania. The number of breeding females is 34000 and breeding
males 1500. Body weight is 65 kg and 50 kg for males and females respectively.
Assessment of genetic variability and genetic characterization of a breed is
very important step for undertaking conservation measurements. The aim of the
present study was the genetic characterization of Muzhake goat breed, using 30
microsatellite markers.

Material and methods

Blood samples were collected
at random from 30 unrelated animals of Muzhake goats. DNA was isolated according
to the standard phenol/chloroform protocol (Sambrook et al 1989). A panel of 30
microsatellite markers as suggested by FAOs MoDAD programme, was used for
genetic characterization of Muzhake goat, according to the methodology explained
in detail by Canon et al 2006).

Microsatellite allele
frequencies, observed and expected number of alleles, effective number of
alleles and Shanon’s Information Index were estimated using Genalex 6 program (Peakall
and Smouse 2006). Observed heterozygosity (Ho), expected heterozygosity (He)
were estimated for 30 microsatellite markers using the same software. Polymorphic
information content (PIC) was estimated for all markers using the Cervus
software (Marshall 1998). Bottleneck events were tested by three methods
implemented in the program BOTTLENECK (Piry et al 1999). Factorial
Correspondence Analysis (FCA) was carried out using GENETIX v4.02 (Belkhir et al
2001).

Ewens-Watterson test was
performed to test the neutrality for microsatellite markers, using the algorithm
by Manly (2007) using 1000 simulated samples and implemented in Popgene software
package (Yeh et al 1999).

Results and discussion

Number of alleles (Na), number
of effective alleles (Ne), expected and unbiased average heterozygosity (He), FIS
and PIC values at each locus are displayed in Table 1. Number of alleles varied
from 4 (ETH10, ILSTS00, MAF209) to 18 (BM6444). There were identified 240
alleles in the whole population for 30 microsatellite markers. Number of
effective alleles varied from 1.47 (MAF209) to 11.5 (BM6444). The PIC values
ranged from 0.30 (MAF209) to 0.91 (BM6444) with a mean value of 0.71. Observed
heterozygosity ranged from 0.33 (MAF209, DRBP1) to 0.93 (OarFCB48), with a mean
value of 0.69. The value of Shannon information index (I) ranged from 0.77 to
2.49. The values of expected heterozygosity ranged from 0.32 (AF209) to 0.91
(BM6444) with a mean value of 0.75. FIS values for all markers ranged
from -0.22 (InraBern185) to 0.589 (DRBP1) with an overall mean estimate of
0.07.

The Chi-square and likelihood
ratio tests were performed to examine Hardy Weinberg Equilibrium (HWE) at each
locus (Table 2). At some loci were revealed significant deviation from HWE (P <
0.05).

The neutrality test of each
marker tested by Ewens-Watterson test for neutrality suggested that most of the
microsatellite loci (Table 2) were neutral and unlinked to any selected trait,
because observed F values lie outside of the upper and lower limits of 95%
confidence region of expected F value. F value (sum of square of allelic
frequency) lay outside the lower and upper limit of 95% confidence region of
expected F value at only BM6444 locus.

Table 3: Bottleneck analysis
for Muzhake goat breed using three different tests under infinite
allele, two phase model and stepwise mutation.

If a population has
experienced a recent bottleneck, rare alleles tend to be lost and the average
number of alleles per locus and gene diversity are reduced. Heterozygosity,
however, is reduced slowly, because rare alleles contribute little to
heterozygosity. Therefore, in a recently bottlenecked population, the observed
gene diversity is higher than the expected equilibrium gene diversity. The
results of three different tests under three microsatellite evolution models for
recent bottleneck are displayed in Table 3. Results of sign test show presence
of bottleneck under TPM, but show the absence of bottleneck under IAM and SMM
model. The sign test revealed significant differences between the number of loci
observed and expected with heterozygosity excess under IAM model (Table 3). 28
loci out of 30 had heterozygosity excess, under IAM and two of loci showed
significant (P <0.01) heterozygosity deficiency. The standardized
difference revealed probability values less than 0.05 for IAM and SMM models.
Therefore the hypothesis of mutation drift equilibrium was not rejected only
under TPM model (T2 = 1.330, P = 0.09183). Wilcoxon test revealed that the
population had undergone recent bottleneck assuming the IAM model. Finally,
mode-shift indicator test was used as a second method to detect potential recent
bottleneck. The microsatellite alleles were organized into 10 frequency classes.
Alleles with low frequencies (0.01 − 0.1) are the most numerous. The
distribution followed the normal L-shaped form (Figure 1) suggesting that the
breed did not encounter a recent genetic bottleneck.

Factorial correspondence
analysis (FCA) suggests that first axis accounted for 5.99%; second 5.76% and
third 5.54% of the total variance (Figure 2). The analysis shows a close genetic
relationship between individuals.

Discussion

Muzhake goat has a high
genetic variation considering gene diversity and average number of alleles per
locus. Gene diversity of Muzhake goat was lower than values found for Raeini
goats (0.805; Sadeghi et al 2010) or Xinong Sanen dairy goat (0.74 – 0.90; Zhu
et al 2011). Number of alleles for each locus was higher than 4.

Average FIS value
is 0.07, indicating a reduction of heterozygotes at 7%. The most probable reason
for this heterozygosity deficiency might be Wahlund effect, since sampling is
carried out in numerous small farms. Since there is lack of herd book for
sampled individuals all information considering nonrelatedness is provided
entirely by the farmers. Therefore sampling of related individuals is not
excluded entirely. This might have contributed in the deficit of heterozygotes.

Conclusion

All microsatellite markers used in the present study were shown to be highly
polymorphic and useful for the molecular characterization of Muzhake
goat breed. The information provided through the present study would be
useful for designing effective conservation strategies.